Cryptococcus neoformans is an opportunistic human pathogen that causes potentially fatal meningoencephalitis in individuals with immune deficiency. Virulence of C. neoformans is a multi-faceted trait with the production of the melanin pigment, a polysaccharide capsule, and secreted proteins contributing to its virulence. The recent introduction of molecular and genetic technologies have allowed us to begin to understand the molecular basis of fungal virulence, yet precision gene alterations and efficient multiplex genome editing that allows systematic examination of virulence mechanisms remain limited due to the lack of advanced approaches. We propose to develop a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system for precise gene specific modification and efficient multiplex genome editing in C. neoformans. The successful development of this innovative genomic approach will provide a revolutionary tool in our studying of virulence mechanisms in C. neoformans.